Oxidative DNA damage accumulates with age and is repaired primarily via the base excision repair (BER) pathway. This process is initiated by DNA glycosylases, which remove damaged bases in a substrate-specific manner. The DNA glycosylases human 8-oxoguanine-DNA glycosylase (OGG1) and NEIL1, a mammalian homolog of Escherichia coli endonuclease VIII, have overlapping yet distinct substrate specificity. Recently, we reported that OGG1 binds to the Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein that poly(ADP-ribosyl)ates nuclear proteins in response to DNA damage and other cellular signals. Here, we show that NEIL1 and PARP-1 bind both in vitro and in vivo. PARP-1 binds to the C-terminal-100 amino acids of NEIL1 and NEIL1 binds to the BRCT domain of PARP-1. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. Furthermore, NEIL-deficient fibroblasts have impaired poly(ADP-ribosyl)ation of cellular proteins after DNA damage, which can be rescued by NEIL1 expression. Additionally, PARP-1 inhibits NEIL1 incision activity in a concentration-dependent manner. Consistent with the idea of impaired DNA repair during aging, we observed differential binding of PARP-1 to recombinant NEIL1 in older mice compared to younger mice. These data further support the idea that dynamic interplay between different base excision repair proteins is important for efficient BER.
PARP; base excision repair; DNA damage; oxidative stress; aging; glycosylase
Persons with elevated blood pressure show dampened emotional responses to affect-laden stimuli. We sought to further examine cardiovascular emotional dampening by examination of the relationship between resting hemodynamic measures and recognition of emotion in an African-American community-based sample.
Participants were 106 African American men and women (55 female; mean age 52.8 years), mainly low in socioeconomic status and part of the Healthy Aging in Nationally Diverse Longitudinal Samples (HANDLS-Pilot) Pilot Study. Participants evaluated emotional expressions in faces and in sentences using the Perception of Affect Test (PAT). Resting blood pressure, total peripheral resistance (TPR), cardiac output and heart rate were obtained continuously using a Portapres blood pressure monitor.
Total PAT scores were inversely related to systolic (r = −.30) and diastolic (r = −.24) blood pressure, TPR (r = −.36) and age (r = − .31; p values < .01), and positively related to cardiac output (r = .27) and education (r = .38; p values <.01), and with mental state (r = .25) and body mass index (r = −.20; p values < .05). Accuracy of emotion recognition on the PAT tasks remained inversely related to TPR and blood pressure after adjustment for demographic variables, medication, mental state and body mass index.
Elevated blood pressure and TPR were associated with reduced perception of affect. TPR was the most consistent independent hemodynamic correlate of emotional dampening for the PAT scores. These results suggest potentially important links among CNS regulation of emotions, hemodynamic processes and hypertension development.
Emotion regulation; blood pressure; hemodynamics; hypertension development; central nervous system; stress
The present study examined the association of serum ferritin with CHD risk using the Framingham Heart Study's 10-year risk algorithm.
Ordinal logistic regression modelling was used to interpret risk. Proportional odds modelling assessed four divisions of ranked CHD risk (4, high; 3, increased; 2, slight; 1, minimal), separately by sex.
Baltimore, MD, USA.
African-American and white participants (n 1823) from baseline of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study, aged 30–64 years.
For men, there was a 0.5% increase in risk for every 10-unit rise in serum ferritin (pmol/l). Other significant predictors included increased BMI, white race, unemployment and C-reactive protein ≥9.5 mg/l. For women, there was a 5.1% increase in risk per 10-unit rise in serum ferritin (pmol/l). Other significant predictors included increased BMI, lower education, unemployment and C-reactive protein ≥9.5 mg/l.
Serum ferritin is a significant predictor of 10-year hard CHD risk for HANDLS study participants, a low-income, urban population. Serum ferritin, independent of elevated C-reactive protein, was associated with increased 10-year CHD risk for HANDLS participants. To our knowledge, these data provide the first evidence of the role of serum ferritin as a risk factor for hard CHD in African-American and white postmenopausal women in the USA. Future research on cardiovascular events from this prospective study may confirm the association.
CVD; Cohort study; Serum ferritin; CHD; Iron
Mutations and polymorphisms of OGG1, the major mammalian 8-oxoguanine repair activity, are associated with increased risk for several cancers. Decreased 8-oxoguanine repair capacity due to variant forms of the OGG1 gene is a common feature of numerous cancer cell lines. One such cell line, human KG-1 leukemia cells, has previously been demonstrated to be deficient in the excision of 8-oxoguanine from oxidatively damaged DNA. KG-1 cells have a homozygous R229Q amino acid substitution in OGG1 that has been presumed to alter the function of OGG1 and result in elevated levels of genomic 8-oxoG and hypersensitivity to 8-hydroxydeoxyguanosine nucleoside and ionizing radiation observed in KG-1 cells.
We characterized the enzymatic activity of R229Q OGG1 and the effect of the enzyme on cell survival following treatment with DNA damaging agents.
R229Q OGG1 had activity similar to the wild-type enzyme, yet was easily heat inactivated at physiological temperature. R229Q OGG1 expressed in human cells had significantly lower activity than wild-type OGG1 and was also highly thermolabile. Expression of R229Q OGG1 sensitized KG-1 cells to killing by menadione and 8-hydroxydeoxyguanosine, but not ionizing radiation.
These results suggest that decreased 8-oxoguanine repair in KG-1 is due to thermolability of R229Q OGG1 and that the enzyme variant increases cellular susceptibility to killing resulting from oxidative DNA damage. The R229Q OGG1 variant is a validated polymorphism prevalent in world populations and not an isolated mutation in KG-1 cells, thus the R229Q OGG1 allele may be a novel marker for cancer susceptibility.
R229Q; OGG1; 8-oxoguanine; KG-1; leukemia; polymorphism
Deficient repair activity for 8-hydroxy-2′-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer’s disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca2+-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (Proline, Glutamic acid, Serine, Threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.
OGG1; calpain; 8-oxoguanine; calcium; PEST
Previous studies indicate a high risk of Posttraumatic Stress Disorder (PTSD) among women and low-income, urban-residing African Americans. This study examined PTSD symptoms among urban-residing, socioeconomically diverse, working age, African Americans and whites. Participants completed the PTSD Checklist-Civilian Version. Of the 2,104 participants, 268 (12.7%) screened positive for PTSD symptoms. Women (13.8%) were more likely than men (11.3%), white participants (13.8%) were more likely than African Americans (11.9%), and younger participants (16.1%) were more likely than older (10.2%) to screen positive for PTSD symptoms. A significant interaction (p = .05) revealed that white women living below the 125% poverty level were most likely to report PTSD symptoms. These findings highlight the importance of PTSD screening in low-income, urban neighborhoods.
Symptoms of Posstraumatic Stress Disorder; civilians; urban residents
Several genetic variants associated with platelet count and mean platelet volume
(MPV) were recently reported in people of European ancestry. In this
meta-analysis of 7 genome-wide association studies (GWAS) enrolling African
Americans, our aim was to identify novel genetic variants associated with
platelet count and MPV. For all cohorts, GWAS analysis was performed using
additive models after adjusting for age, sex, and population stratification. For
both platelet phenotypes, meta-analyses were conducted using inverse-variance
weighted fixed-effect models. Platelet aggregation assays in whole blood were
performed in the participants of the GeneSTAR cohort. Genetic variants in ten
independent regions were associated with platelet count
(N = 16,388) with p<5×10−8 of
which 5 have not been associated with platelet count in previous GWAS. The novel
genetic variants associated with platelet count were in the following regions
(the most significant SNP, closest gene, and p-value): 6p22 (rs12526480,
LRRC16A, p = 9.1×10−9), 7q11
(rs13236689, CD36, p = 2.8×10−9),
10q21 (rs7896518, JMJD1C,
p = 2.3×10−12), 11q13 (rs477895,
BAD, p = 4.9×10−8), and 20q13
(rs151361, SLMO2, p = 9.4×10−9).
Three of these loci (10q21, 11q13, and 20q13) were replicated in European
Americans (N = 14,909) and one (11q13) in Hispanic
Americans (N = 3,462). For MPV
(N = 4,531), genetic variants in 3 regions were significant
at p<5×10−8, two of which were also associated with
platelet count. Previously reported regions that were also significant in this
study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22,
17q11, and 19p13 for MPV. The most significant SNP in 1 region was also
associated with ADP-induced maximal platelet aggregation in whole blood (12q24).
Thus through a meta-analysis of GWAS enrolling African Americans, we have
identified 5 novel regions associated with platelet count of which 3 were
replicated in other ethnic groups. In addition, we also found one region
associated with platelet aggregation that may play a potential role in
The majority of the variation in platelet count and mean platelet volume between
individuals is heritable. We performed genome-wide association studies in more
than 16,000 African American participants from seven population-based cohorts to
identify genetic variants that correlate with variation in platelet count and
mean platelet volume. We observed statistically significant evidence
(p-value<5×10−8) that 10 genomic regions were
associated with platelet count and 3 were associated with mean platelet volume.
Of the regions that were significantly associated, we found 5 novel regions that
were not reported previously in other populations. Three of these 5 regions were
also associated with platelet count in European Americans and Hispanic
Americans. All these regions contain genes that are either known to have or
potentially may have a role in determining platelet count and/or mean platelet
volume. We further found that one of these regions was also associated with
agonist-induced platelet aggregation. Further studies will determine the exact
role played by these genomic regions in platelet biology. The knowledge
generated by this and other studies will not only help us better understand
platelet biology but can also lead us to the discovery of new anti-platelet
To assess the predictive values of various adiposity indices for the metabolic syndrome (MetS) among adults using baseline data from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) cohort.
In a cross-sectional study, body mass index (BMI), waist circumference (WC), body composition by dual-energy x-ray absorptiometry (DXA) and metabolic risk factors such as triglycerides, HDL-cholesterol, blood pressure, fasting glucose and insulin, uric acid and C-reactive protein were measured. Receiver-operating characteristic (ROC) curves and logistic regression analyses were conducted.
1,981 White and African-American US adults, aged 30-64 years.
In predicting risk of MetS using obesity-independent National Cholesterol Education Program Adult Treatment Panel III criteria, % body fat mass (TtFM) assessed using DXA measuring overall adiposity had no added value over WC. This was true among both men (Areas under curve; AUC=0.680 vs. 0.733 for TtFM and WC, respectively, p<0.05) and women (AUC=0.581 vs. 0.686). Rib fat mass (RbFM) was superior to TtFM only in women for MetS (AUC=0.701 and 0.581 for RbFM and TtFM, respectively, p<0.05), particularly among African-American women. Elevated Leg fat mass (LgFM) was protective against MetS among African-American men. Among White men, BMI was inferior to WC in predicting MetS. Optimal WC cut points varied across ethnic-sex groups and differed from those recommended by the NIH/NAASO.
We provide evidence that WC is among the most powerful tools to predict MetS, and that optimal cut-points for various indices including WC may differ by sex and race.
Metabolic syndrome; percent body fat mass; central obesity; body mass index
Low socioeconomic status (SES) and African American race are both independently associated with end-stage renal disease and progressive chronic kidney disease (CKD), however, despite their frequent co-occurrence, the effect of low SES independent of race has not been well-studied in CKD.
Setting & Participants
2,375 community-dwelling adults age 30-64 years residing within 12 neighborhoods selected for both socioeconomic and racial diversity in Baltimore City, Maryland.
Low SES [self-reported household income <125% of 2004 Department of Health and Human Services guideline], higher SES (≥125% of guideline); white and African American race.
Outcomes & Measurements
CKD defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Logistic regression used to calculate odds ratios (OR) for relationship between poverty and CKD, stratified by race.
Of 2,375 participants; 955 were white (347 low SES and 608 higher SES); 1,420 were African American (713 low SES and 707 higher SES). A total of 146 (6.2%) participants had CKD. Overall, race was not associated with CKD [OR, 1.05; 95% confidence interval (CI), 0.57-1.96]; however, African Americans had a much greater odds of advanced CKD (eGFR <30 mL/min/1.73 m2). Low SES was independently associated with 59% greater odds of CKD after adjustment for demographics, insurance status and comorbid disease (OR, 1.59; 95% CI, 1.27-1.99). However, when stratified by race, low SES was associated with CKD in African Americans (OR, 1.91; 95% CI, 1.54-2.38), but not in whites (OR, 0.95; 95% CI, 0.58-1.55; P for interaction, 0.003).
Cross-sectional design; findings may not be generalizable to non-urban populations.
Low SES has a profound relationship with CKD in African Americans but not in whites in an urban population of adults, and its role in the racial disparities seen in CKD is worthy of further investigation.
Socioeconomic status; health disparities; epidemiology; renal disease
Previous literature has shown that the availability of healthy food in neighborhoods is associated with area characteristics and dietary quality. This study investigated the association between the availability of healthy foods and body mass index (BMI) among 2,616 participants living in Baltimore City.
Trained staff collected demographic information, height, weight and 24-hr dietary recalls between 2004 and 2008. Healthy food availability was determined in 34 census tracts of varying racial and SES composition using the Nutrition Environment Measures Survey- Stores in 2007. Multilevel linear regression was used to estimate associations between healthy food availability and BMI.
Among individuals living in predominately white neighborhoods, high availability of healthy foods was associated with significantly higher BMI compared to individuals living in low healthy food availability neighborhoods after adjustment for demographic variables (β=3.22, p=0.001). Associations were attenuated but remained significant after controlling for dietary intake (β=2.81, p=0.012).
Contrary to expectations, there was a positive association between the availability of healthy food and higher BMI among individuals living in predominately white neighborhoods. This result could be due to individuals in low healthy food availability areas traveling outside their neighborhood to obtain healthy food.
Body mass index; Healthy Food Availability; Obesity; BMI; Neighborhood; Racial Composition; Socioeconomic Status
Purpose of the study: Investigating health disparities requires studies designed to recruit and retain racially and socioeconomically diverse cohorts. It is critical to address the barriers that disproportionately affect participation in clinical research by minorities and the socioeconomically disadvantaged. This study sought to identify and rectify these barriers to recruit and retain a biracial (African American and non-Hispanic White) and socioeconomically diverse cohort for a longitudinal study. Design and Method: The Healthy Aging in Neighborhoods of Diversity across the Life Span study is a 20-year longitudinal examination of how race and socioeconomic status influence the development of age-related health disparities. One goal was to create a multifactorial recruitment and retention strategy. The recruitment paradigm targeted known barriers and identified those unique to the study's urban environment. The retention paradigm mirrored the recruitment plan but was based on specifically developed approaches. Results: This cohort recruitment required attention to developing community partnerships, designing the research study to meet the study hypotheses and to provide benefit to participants, providing a safe community-based site for the research and creating didactics to develop staff cultural proficiency. These efforts facilitated study implementation and enhanced recruitment resulting in accrual of a biracial and socioeconomically diverse cohort of 3,722 participants. Implications: Recruiting and retaining minority or poor research participants is challenging but possible. The essential facets include clear communication of the research hypothesis, focus on providing a direct benefit for participants, and selection of a hypothesis that is directly relevant to the community studied
Cultural proficiency; Health disparities; Community-based research platform
Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4, dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive ELISA methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan study, oxidative stress in humans were quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls that were both different from the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R=0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.
Protein carbonyl; fluorescence; fluorimetric; assay; human plasma; fluorescein thiosemicarbazide; micro plate format
Recently, we developed an improved comet assay protocol for evaluating single-strand break repair capacity (SSB-RC) in unstimulated cryopreserved human peripheral blood mononuclear cells (PBMCs). This methodology facilitates control of interexperimental variability [A.R. Trzeciak, J. Barnes, M.K. Evans, A modified alkaline comet assay for measuring DNA repair capacity in human populations. Radiat. Res. 169 (2008) 110-121]. The fast component of SSB repair (F-SSB-RC) was assessed using a novel parameter, the initial rate of DNA repair, and the widely used half-time of DNA repair. The slow component of SSB repair (SSSB-RC) was estimated using the residual DNA damage after 60 min. We have examined repair of γ-radiation-induced DNA damage in PBMCs from four age-matched groups of male and female whites and African-Americans between ages 30 and 64. There is an increase in F-SSB-RC with age in white females (P<0.01) and nonsignificant decrease in F-SSB-RC in African-American females (P=0.061). F-SSB-RC is lower in white females than in white males (P<0.01). There is a decrease in F-SSB-RC with age in African-American females as compared to white females (P<0.002) and African-American males (nonsignificant, P=0.059). Age, sex, and race had a similar effect on intercellular variability of DNA damage in γ-irradiated and repairing PBMCs. Our findings suggest that age, sex, and race influence SSB-RC as measured by the alkaline comet assay. SSB-RC may be a useful clinical biomarker.
SSB repair capacity; SSB repair kinetics; DNA repair; Comet assay; γ-irradiation; Cryopreserved lymphocytes; Oxidative DNA damage; Reference standards; Biomonitoring study; Dispersion coefficient H; Coefficient of variation
Examine the influences of race, socioeconomic status, sex, and age on barriers to participation in a study of cross-sectional differences and longitudinal changes in health-related outcomes.
We designed a multidisciplinary, community-based, prospective longitudinal epidemiologic study among socioeconomically diverse African Americans and whites. The study protocol facilitated our ability to recruit 3722 participants from Baltimore, MD with mean age 47.7 (range 30–64) years, % males/female, 2200 African Americans (59%) and 1522 whites (41%); 41% reported household incomes below the 125% poverty delimiter.
There were no significant age differences associated with sex or race. Participants below the 125% poverty delimiter were slightly younger than those above the delimiter. Age, race, and sex, but not poverty status, were associated with the likelihood of an examination. Older participants, women, and whites were more likely to complete their examinations. Among those who completed their examinations, there were no age differences associated with sex and poverty status, but African Americans were negligibly younger than whites.
Although some literature suggests that minorities and low-income people are less willing to participate in clinical research, these baseline data suggest that African Americans individuals and individuals from households with incomes below 125% of the poverty level are at least as willing to participate in observational clinical studies as whites and higher income individuals of similar age and sex.
Healthcare disparities; socioeconomic status; Population groups; Epidemiologic research design; Health surveys; Longitudinal studies
To examine effects of race and predictors of socioeconomic status (SES) on nutrient-based diet quality and their contribution to health disparities in an urban population of low SES.
Data were analyzed from a sample of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) Study participants examining effects of age, sex, race, income, poverty income ratio (PIR), education, employment, and smoking status on nutrient-based diet quality as measured by a micronutrient composite index of nutrient adequacy ratios (NAR) and a mean adequacy ratio (MAR). Regression models were used to examine associations and t-tests were used to look at racial differences.
African American and white adults ages 30-64 residing in 12 predefined census tracts in Baltimore City, Maryland.
Sex, age, education, PIR, and income were statistically significant predictors of diet quality for African Americans, while sex, education, and smoking status were statistically significant for whites. African Americans had lower MAR scores than whites (76.4 vs. 79.1). Whites had significantly higher NAR scores for thiamin, riboflavin, folate, B12, vitamins A and E, magnesium, copper, zinc, and calcium, while African Americans had higher vitamin C scores.
Education significantly impacted diet quality in the HANDLS sample, but race cannot be discounted. Whether the racial differences in diet quality are indicative of cultural differences in food preferences, selection, preparation, and availability or disparities in socioeconomic status remains unclear.
Race; Socioeconomic Status; Education; Diet Quality
High-energy-dense foods provide an inexpensive source of calories. Healthy Aging in Neighborhoods of Diversity across the Life Span study participants (n = 1987), low- to low-middle-income, urban African American and white adults, consumed between 17% and 20% of their daily energy intake from beverages. Of all beverages consumed, calorically sweetened beverages ranked second among African Americans and third among whites. Calorically-sweetened beverage consumption was not influenced by weight status. Increasing awareness of risks for adverse health outcomes associated with selected beverages may improve dietary choices.
energy intake; obesity
Previous research has shown that reading ability is a stronger predictor of cognitive functioning than years of education, particularly for African Americans. The current study was designed to determine whether the relative influence of literacy and education on cognitive abilities varies as a function of race or socioeconomic status (SES). We examined the unique influence of education and reading scores on a range of cognitive tests in low and high SES African Americans and Whites. Literacy significantly predicted scores on all but one cognitive measure in both African American groups and in low SES Whites, while education was not significantly associated with any cognitive measure. In contrast, both education and reading scores predicted performance on many cognitive measures in high SES Whites. These findings provide further evidence that reading ability better predicts cognitive functioning than years of education and suggest that disadvantages associated with racial minority status and low SES affect the relative influence of literacy and years of education on cognition.
reading; income; demographics; ethnicity; neuropsychological testing; cognition
Recent evidence supports a role for microRNAs (miRNAs) in regulating the life span of model organisms. However, little is known about how these small RNAs contribute to human aging. Here, we profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.
Elevated levels of oxidatively induced DNA lesions have been reported in malignant pancreatic tissues relative to normal pancreatic tissues. However, the ability of the pancreatic cancer cells to remove these lesions has not previously been addressed. This study analyzed the effectiveness of the pancreatic cancer cell line, BxPC-3 to repair 8-hydroxyguanine (8-OH-Gua) relative to a nonmalignant cell line. We show that BxPC-3 cells repair 8-OH-Gua less effectively than the nonmalignant cells. This repair deficiency correlated with significant downregulation of the hOGG1 protein and the corresponding mRNA (30-fold lower than GAPDH) in BxPC-3 cell line. The repair defect was complemented in vivo by transient transfection of the hOGG1 gene and in vivo by recombinant hOGG1. These results are the first to show a deficiency of 8-OH-Gua repair in BxPC-3 cells, implicating this defect in the risk factor of pancreatic cancer.
Pancreatic cancer; Oxidative stress; Oxidative DNA damage; DNA repair; Base excision repair; Reactive oxygen species; 8-Hydroxyguanine; hOGG1; Pancreatic cancer risk and antioxidant; enzymes
emotion recognition; personality traits; openness; sex differences; cross; cultural
Breast cancer is the second leading cause of cancer deaths in women in the United States. Although the causes of this disease are incompletely understood, oxidative DNA damage is presumed to play a critical role in breast carcinogenesis. A common oxidatively induced DNA lesion is 8-hydroxyguanine (8-OH-Gua), which has been implicated in carcinogenesis. The aim of this study was to investigate the ability of HCC1937 and MCF-7 breast cancer cell lines to repair 8-OH-Gua relative to a nonmalignant human mammary epithelial cell line, AG11134.
We used oligonucleotide incision assay to analyze the ability of the two breast cancer cell lines to incise 8-OH-Gua relative to the control cell line. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the levels of 8-OH-Gua as its nucleoside, 8-OH-dG in the cell lines after exposure to H2O2 followed by 30 min repair period. Protein expression levels were determined by Western blot analysis, while the hOGG1 mRNA levels were analyzed by RT-PCR. Complementation of hOGG1 activity in HCC1937 cells was assessed by addition of the purified protein in the incision assay, and in vivo by transfection of pFlagCMV-4-hOGG1. Clonogenic survival assay was used to determine sensitivity after H2O2-mediated oxidative stress.
We show that the HCC1937 breast cancer cells have diminished ability to incise 8-OH-Gua and they accumulate higher levels of 8-OH-dG in the nuclear genome after H2O2 treatment despite a 30 min repair period when compared to the nonmalignant mammary cells. The defective incision of 8-OH-Gua was consistent with expression of undetectable amounts of hOGG1 in HCC1937 cells. The reduced incision activity was significantly stimulated by addition of purified hOGG1. Furthermore, transfection of pFlagCMV-4-hOGG1 in HCC1937 cells resulted in enhanced incision of 8-OH-Gua. HCC1937 cells are more sensitive to high levels of H2O2 and have up-regulated SOD1 and SOD2.
This study provides evidence for inefficient repair of 8-OH-Gua in HCC1937 breast cancer cell line and directly implicates hOGG1 in this defect.
Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.
Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.
Meta-analyses of European populations has successfully identified genetic variants in over 100 loci associated with lipid levels, but our knowledge in other ethnicities remains limited. To address this, we performed dense genotyping of ∼2,000 candidate genes in 7,657 African Americans, 1,315 Hispanics and 841 East Asians, using the IBC array, a custom ∼50,000 SNP genotyping array. Meta-analyses confirmed 16 lipid loci previously established in European populations at genome-wide significance level, and found multiple independent association signals within these lipid loci. Initial discovery and in silico follow-up in 7,000 additional African American samples, confirmed two novel loci: rs5030359 within ICAM1 is associated with total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (p = 8.8×10−7 and p = 1.5×10−6 respectively) and a nonsense mutation rs3211938 within CD36 is associated with high-density lipoprotein cholesterol (HDL-C) levels (p = 13.5×10−12). The rs3211938-G allele, which is nearly absent in European and Asian populations, has been previously found to be associated with CD36 deficiency and shows a signature of selection in Africans and African Americans. Finally, we have evaluated the effect of SNPs established in European populations on lipid levels in multi-ethnic populations and show that most known lipid association signals span across ethnicities. However, differences between populations, especially differences in allele frequency, can be leveraged to identify novel signals, as shown by the discovery of ICAM1 and CD36 in the current report.
Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using 14C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10−8): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10−9), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10−32) and SLC22A12 (P= 2.1 × 10−10). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta −1.19 mg/dl, P= 2.7 × 10−16). 14C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.